Multiple research groups have shown that diet impacts the gut microbiome; however, variability in experimental design and quantitative assessment have made it challenging to assess the degree to which similar diets have reproducible effects across studies. Through an unbiased subject-level meta-analysis framework, we re-analyzed 27 dietary studies including 1,101 samples from rodents and humans. We demonstrate that a high-fat diet (HFD) reproducibly changes gut microbial community structure. Finer taxonomic analysis revealed that the most reproducible signals of a HFD are Lactococcus species, which we experimentally demonstrate to be common dietary contaminants. Additionally, a machine-learning approach defined a signature that predicts the dietary intake of mice and demonstrated that phylogenetic and gene-centric transformations of this model can be translated to humans. Together, these results demonstrate the utility of microbiome meta-analyses in identifying robust and reproducible features for mechanistic studies in preclinical models.

Very low-carbohydrate, high-fat ketogenic diets (KDs) induce a pronounced shift in metabolic fuel utilization that elevates circulating ketone bodies; however, the consequences of these compounds for host-microbiome interactions remain unknown. Here, we show that KDs alter the human and mouse gut microbiota in a manner distinct from high-fat diets (HFDs). Metagenomic and metabolomic analyses of stool samples from an 8-week inpatient study revealed marked shifts in gut microbial community structure and function during the KD. Gradient diet experiments in mice confirmed the unique impact of KDs relative to HFDs with a reproducible depletion of bifidobacteria. In vitro and in vivo experiments showed that ketone bodies selectively inhibited bifidobacterial growth. Finally, mono-colonizations and human microbiome transplantations into germ-free mice revealed that the KD-associated gut microbiota reduces the levels of intestinal pro-inflammatory Th17 cells. Together, these results highlight the importance of trans-kingdom chemical dialogs for mediating the host response to dietary interventions.

A20 is an anti-inflammatory protein that is strongly linked to human disease. Here, we find that mice expressing three distinct targeted mutations of A20's zinc finger 7 (ZF7) ubiquitin-binding motif uniformly developed digit arthritis with features common to psoriatic arthritis, while mice expressing point mutations in A20's OTU or ZF4 motifs did not exhibit this phenotype. Arthritis in A20^ZF7 mice required T cells and MyD88, was exquisitely sensitive to tumor necrosis factor and interleukin-17A, and persisted in germ-free conditions. A20^ZF7 cells exhibited prolonged IκB kinase activity that drove exaggerated transcription of late-phase nuclear factor-κB response genes in vitro and in prediseased mouse paws in vivo. In addition, mice expressing double-mutant A20 proteins in A20's ZF4 and ZF7 motifs died perinatally with multi-organ inflammation. Therefore, A20's ZF4 and ZF7 motifs synergistically prevent inflammatory disease in a non-catalytic manner.

Diet can protect from autoimmune disease; however, whether diet acts via the host and/or microbiome remains unclear. Here, we use a ketogenic diet (KD) as a model to dissect these complex interactions. A KD rescued the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis in a microbiota-dependent fashion. Dietary supplementation with a single KD-dependent host metabolite (β-hydroxybutyrate [βHB]) rescued EAE, whereas transgenic mice unable to produce βHB in the intestine developed more severe disease. Transplantation of the βHB-shaped gut microbiota was protective. Lactobacillus sequence variants were associated with decreased T helper 17 cell activation in vitro. Finally, we isolated an L. murinus strain that protected from EAE, which was phenocopied by a Lactobacillus metabolite enriched by βHB supplementation, indole lactate. Thus, diet alters the immunomodulatory potential of the gut microbiota by shifting host metabolism, emphasizing the utility of taking a more integrative approach to study diet-host-microbiome interactions.

Diet is a major factor that shapes the gut microbiome¹, but the consequences of diet-induced changes in the microbiome for host pathophysiology remain poorly understood. We conducted a randomized human intervention study using a very-low-calorie diet (NCT01105143). Although metabolic health was improved, severe calorie restriction led to a decrease in bacterial abundance and restructuring of the gut microbiome. Transplantation of post-diet microbiota to mice decreased their body weight and adiposity relative to mice that received pre-diet microbiota. Weight loss was associated with impaired nutrient absorption and enrichment in Clostridioides difficile, which was consistent with a decrease in bile acids and was sufficient to replicate metabolic phenotypes in mice in a toxin-dependent manner. These results emphasize the importance of diet-microbiome interactions in modulating host energy balance and the need to understand the role of diet in the interplay between pathogenic and beneficial symbionts.

Anti-TNF antibodies are effective for treating patients with inflammatory bowel disease (IBD), but many patients fail to respond to anti-TNF therapy, highlighting the importance of TNF-independent disease. We previously demonstrated that acute deletion of 2 IBD susceptibility genes, A20 (Tnfaip3) and Abin-1 (Tnip1), in intestinal epithelial cells (IECs) sensitized mice to both TNF-dependent and TNF-independent death. Here we show that TNF-independent IEC death after A20 and Abin-1 deletion was rescued by germ-free derivation or deletion of MyD88, while deletion of Trif provided only partial protection. Combined deletion of Ripk3 and Casp8, which inhibits both apoptotic and necroptotic death, completely protected against death after acute deletion of A20 and Abin-1 in IECs. A20- and Abin-1-deficient IECs were sensitized to TNF-independent, TNFR1-mediated death in response to lymphotoxin α (LTα) homotrimers. Blockade of LTα in vivo reduced weight loss and improved survival when combined with partial deletion of MyD88. Biopsies of inflamed colon mucosa from patients with IBD exhibited increased LTA and IL1B expression, including a subset of patients with active colitis on anti-TNF therapy. These data show that microbial signals, MyD88, and LTα all contribute to TNF-independent intestinal injury.

Dose-limiting toxicities remain a major barrier to drug development and therapy, revealing the limited predictive power of human genetics. Here, we demonstrate the utility of a more comprehensive approach to studying drug toxicity through longitudinal profiling of the human gut microbiome during colorectal cancer (CRC) treatment (NCT04054908) coupled to cell culture and mouse experiments. Substantial shifts in gut microbial community structure during oral fluoropyrimidine treatment across multiple patient cohorts, in mouse small and large intestinal contents, and in patient-derived ex vivo communities were revealed by 16S rRNA gene sequencing. Metagenomic sequencing revealed marked shifts in pyrimidine-related gene abundance during oral fluoropyrimidine treatment, including enrichment of the preTA operon, which was sufficient for the inactivation of active metabolite 5-fluorouracil (5-FU). preTA+ bacteria depleted 5-FU in gut microbiota grown ex vivo and in the mouse distal gut. Germ-free and antibiotic-treated mice experienced increased fluoropyrimidine toxicity, which was rescued by colonization with the mouse gut microbiota, preTA+ Escherichia coli, or preTA-high stool from patients with CRC. Last, preTA abundance was negatively associated with fluoropyrimidine toxicity in patients. Together, these data support a causal, clinically relevant interaction between a human gut bacterial operon and the dose-limiting side effects of cancer treatment. Our approach may be generalizable to other drugs, including cancer immunotherapies, and provides valuable insights into host-microbiome interactions in the context of disease.